Directed Evolution of Microcin J25 Towards Potency Against Resistant Escherichia coli

Author/​Artist
Zah, Eugenia [Browse]
Format
Senior thesis
Language
English
Description
52 pages

Details

Advisor(s)
Link, A. James [Browse]
Department
Princeton University. Department of Chemical and Biological Engineering [Browse]
Class year
2013
Restrictions note
Walk-in Access. This thesis can only be viewed on computer terminals at the Mudd Manuscript Library.
Summary note
Microcin J25 (MccJ25) is a 21 amino acid antibacterial peptide whose lasso structure endows it with increased stability and resistance to many proteases. Capable of killing Gram-negative enterobacteria such as Escherichia coli and Salmonella newport, MccJ25 is a strong candidate for protein antibiotics. However, for MccJ25 to remain a promising antibiotic candidate, it must be shown that improved functionalities can be incorporated into the peptide even against a resistant strain. Previously, a strain of E. coli carrying the Thr931-Ile mutation in the G-G' section of the rpoC gene that encodes for the β' subunit of RNA polymerase (RNAP) was shown to be resistant to wild-type MccJ25. In addition, previous work demonstrated that incorporation of mutations at up to three positions in the peptide can produce active MccJ25 variants, some even more active than wild-type. In this work, a MccJ25-resistant strain of E. coli was generated by incorporating the mutation for resistance into the bacterial genome using genetic recombination via the Wanner method. Then, libraries of MccJ25 variants were screened against the resistant strain to select for mutants capable of overcoming the bacterial resistance. Showing that MccJ25 can be evolved to overcome a resistant strain would prove that natural evolution that bacteria have evolved for survival can be countered with laboratory directed evolution.

Supplementary Information